A problem with using E. coli and other prokaryotic microorganisms as hosts for expression of desired proteins has often been externalizing the proteins from the host cells for purification. Attempts to overcome this problem include physical disruption of cells such as by homogenization or sonication, chemical disruption of cells such as by treatment with detergent or lysozyme, and fusing a DNA sequence which codes for an excretion signal peptide to a structural gene coding for the desired product. For example, Weissman et al., European Patent Application No. 61,250, disclose treatment of host cells with a lysing or permeabilizing agent; Silhavy et al., U.S. Pat. No. 4,336,336, disclose a method for fusing a gene for a cytoplasmic protein to a gene for a non-cytoplasmic protein, so that a resulting hybrid protein is transported to, near or beyond the host cell surface; Gilbert et al., U.S. Pat. No. 4,338,397, disclose a method for producing mature secreted proteins comprising inserting a structural gene for a preprotein into an expression vector.
E. coli can be infected by an obligatory parasite, the lambda phage, which is a double-stranded DNA virus. Lambda genetics, like E. coli genetics, is well-studied. See, for example, "The Bacteriophage Lambda," edit. by A. D. Hershey, Cold Spring Harbor Laboratory, New York, 1971.
Lambda, a temperate phage, multiplies in E. coli in either of two phases. In one, the lytic phase, the phage DNA replicates autonomously and directs formation of capsid proteins, packaging and host cell lysis. Expression of lambda DNA during the lytic phase is highly efficient. Transcription occurs on both DNA strands, on one in the rightward direction and on the other in the leftward direction. Induction can result in release of about one hundred phage particles within 50 minutes at 37.degree. C. See, Hershey, above.
In the other phase, the lysogenic phase, lambda DNA is integrated into the host cell genome and is replicated, passively, along with the host chromosomal DNA by the host replication enzymes. A phage in the lysogenic phase is known as a prophage; the host is known as a lysogen and is said to be immune.
Immunity can be lost by occurrence of various events which induce the lytic phase. The products of the lambda int and xis genes catalyse excision of the lambda genome from the E. coli genome to form a covalently closed circle capable of autonomous replication. The synthesis of these genes, and either directly or indirectly, all other lambda genes is repressed by the product of the lambda cI gene. In response to certain chemicals or DNA-damaging agents, the bacteria directs synthesis of the product of the bacterial recA gene. The recA gene product proteolytically cleaves the cI repressor protein, permitting expression of the lytic phase genes. Propagation of the phage then requires interplay of several lambda regulatory elements which ultimately initiate autonomous replication of the lambda DNA. The products of the lambda P and O genes are required for DNA replication. Subsequent to DNA replication the phage must direct synthesis of viral structural proteins, that is, head and tail proteins, and their assembly into intact empty virions. Interaction of at least 18 genes is required to accomplish this. Finally, the DNA is packaged into the empty virions to produce infectious intact virions, and the cell is ruptured by endolysin, coded for by the lambda S and R genes which are activated by the product of the Q gene, thereby releasing the phages. The Q gene is activated by the N function. The N gene is repressed by the cI function.
The 18 genes required for capsid assembly lie between about map positions 3 and 36 on the rightward transcription strand, map positions being representative of percentages of total lambda DNA. The first genes, from left to right, are A, W and B; the last is J. In normal lysogens, to the right of the J gene are eight bacterial genes. Five of these, bio A, B, C, D and F, are involved in biosynthesis of biotin. A sixth, uvrB, confers resistance to ultraviolet radiation. The final two, chlA and E, confer sensitivity to chlorates. See, Guest, Mol. Gen. Genet. 105: 285-289 (1969) and Stevens et al, in "The Bacteriophage Lambda", ed. by Hershey, et al, cited above, at pp. 515-534.
Another lambda gene which functions in natural host cell lysis is the kil gene. The function of the kil gene is not fully understood. Cells which express the kil gene have a decreased rate of cell growth following induction. Loss of the kil function permits cells to grow at normal rates, that is, log phase growth, after induction, until lysis occurs. Like the S and R genes, the kil gene is regulated by the cI repressor, indirectly, through the N gene. See, Greer, Virology 66:589-604 (1975).
Temperature sensitive lysogens have been well-studied. They are described, for example, by Campbell, Virology 14: 22-32 (1961). The cI857 gene is a temperature sensitive cI mutant. It is functional at or below 38.degree. C. See, Sussman et al., C.R.H. Acad. Sci. Paris 254:1517-1519 (1962). Similar phage systems are known to occur in other genera. For example, Lomovskaya et al., J. Virol. 9:258-262 (1972), report temperature sensitive mutants of a temperate phage which infects Streptomyces; Flock, Mol. Gen. Genet. 155: 241-247 (1977), reports temperature sensitive mutants of the temperate phage, phi-105, which infects Bacillus; Botstein et al., Nature 251: 584-588 (1974) report temperature sensitive mutants of the temperate phage, P22, which infects Salmonella. Jostrom et al., J. Bacteriol. 119:19-32 (1974), and Thompson, J. Bacteriol. 129:778-788 (1977), report temperature sensitive mutants of the temperate phage, phi-11, which infects Staphylococcus; Miller et al, Virol. 59:566-569 (1974) report temperate phages of Pseudomonas.
The lambda endolysin has been found to lyse Salmonella strains which are able to absorb the phage as reported by Botstein et al., Ann. Rev. Genetics 16:61-83 (1982).
Perricaudet et al., FEBS Lett. 56:7-11 (1975), describe deletion of lambda genes between map positions 58 and 71 (.DELTA. 58-71) which segment includes the lambda int, xis, red, gam, cIII and kil genes.
Hershberger et al., United Kingdom Specification Application No. 2,084,584, disclose use of a lysogen as a host cell to stabilize and select for the presence of a plasmid. The authors disclose, for example, transforming a lysogen having a defective cI gene with a plasmid carrying a functional cI gene. In one disclosed embodiment, the functional cI gene is the cI857 gene.
It is known that transposable elements, that is, genes which can recombine independently of host chromosomal recombination mechanisms, can be inserted into host cells as markers. Ross et al., Cell 16:721-731 (1979), report physical structures of deletions and inversions promoted by the transposable tetracycline-resistance element, tn10. Davis et al., "Bacterial Genetics", Cold Spring Harbor Laboratory, New York (1980), describe uses of transposable elements.
Ruvkun et al., Nature 289:85-88 (1981), report integration of the transposable kanamycin resistance and neomycin resistance element, tn5, into Rhizobium meliloti chromosomal DNA by conjugation of a plasmid carrying tn5 followed by homologous recombination. Integration of a heterologous gene by recombination resulting from presence of homologous flanking sequences is also disclosed in European Patent Application No. 74,808.